ZOMATION AND DIFFERENTIATION OF TISSUES IN THE
PRIMARY ROOT OF SOYBEAN
DISSERTATION
Presented in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy in the
Graduate School of The Ohio State
University
3 y
CHAO NIEN SUN, B. Sc., M. Sc.
The Ohio State University
1953
Approved by:
Adviser {/ ACKNOWLEDGEMENT
This investigation was carried out under the direction of Dr.
R. A. Popham of the Department of Botany and Plant Pathology, The
Ohio State University, Columbus, Ohio. The writer wishes to express his deep gratitude to him and also to all who have aided in any way during the course of this study and in the preparation of this paper.
i A Q 9 8 4 3 TABLE OF CONTENTS
I. ORGANIZATION OF THE ROOT APICAL MERISTEM
INTRODUCTION ...... 1
MATERIALS AND METHODS ...... k
GENERAL PATTERN OF ZONATION ...... 7
1. The stelar initials and their derivatives ...... 7
2. The common initials and their derivatives ...... 8
3* Comparison of apices of primary roots of various
ages ...... 9
DISCUSSION ...... 16
SUMMARY ...... 18
LITERATURE CITED ...... 19
II. GROWTH AND TISSUE DIFFERENTIATION IN PRIMARY ROOTS ...... 21
PROCEDURES .... 22
EXPERIMENTAL RESULTS ...... 23
1. External morphology...... 23
2. General structure of the primary root ...... 25
3- Tissue differentiation...... 27
A. Primary phloem ...... 27
B. Primary xylem ...... 28
C. Stelar' cambium ...... 3h
D . Pericycle ...... R2
E. Pericycle - secondary root origin ...... 1x3
F. Endodermis ...... U3
G. Cortical parenchyma ...... AA
H. Epidermis and root hairs ...... ilA
ii I. Secondary tissues ...... 1+5
J. Root cap ...... 1+5
K. Comparison of growth region and tissue
differentiation in aerated and non-aerated
roots ...... 1+6
DISCUSSION ...... 1+7
SUMMARY...... h9
LITERATURE CITED ...... 51
AUTOBIOGRAPHY ...... 53
iii ZONATION AND DIFFERENTIATION OF TISSUES IN THE
PRIMARY ROOT OF SOYBEAN
I. ORGANIZATION OF THE ROOT APICAL MERISTEM
INTRODUCTION
Much work has been done within the last decade on zonation
structure of shoot apices, but recent investigations on root apical meristems are surprisingly few. The present study undertakes to
clarify the details of zonation in the soybean root tip.
Perhaps the earliest study of the root apex is that by NM-geli
(18145)* Some 25 years later, Hanstein (1870) founded the well known histogen theory of apical meristem organization. Although his concept
is not applicable to shoot meristems, it is still useful in describing
the organization and origin of root tissues. Later workers, such as * Janczewski (l87li)> Eriksson (1878), and Flahaut (1878), classified
root meristems according to their interpretation of the histogen theory.
Because soybean belongs to Leguminosae, a brief outline of the
structural differences among root apices of Leguminosae studied and recorded to date will be presented. Janczewski (I87I4) described five types of promeristems for the phanerogams. In his fourth type, for which the roots of some Leguminosae (Pisurn sativum and Phaseplus vulgaris) furnish examples, all tissues (the central cylinder, cortex,
epidermis, and root cap) originate from a common meristematic zone.
Eriksson (I878) distinguished four types of root apices for dicotyledons. His third type corresponds to Janczewski*s fourth type, being characterized by the fact that all of the primary tissues of the root originate from a common meristematic zone. In this class he places 2
the following species of Leguminosae: Vicia sativa, V. narbonensis,
Pisum sativum, Cicer arietinum, Phaseplus multiflorus, Lathyrus
odoratus, L. latifolius, Robinia pseudacacia, and Cassia glauca.
Eriksson's fourth type, on the other hand, is characterized by the
presence, at the apex of the root, of two different meristems, a
plerome and a group of common initials. The former gives rise to the
central cylinder. The latter gives rise to the cortex, epidermis and
root cap. The root cap consists of two sharply defined parts: the
columella and the peripheral portion. The columella is derived from
common initials by transverse cell divisions while the peripheral part
of the root cap is derived from the common initials by tangential cell
divisions. In this group he placed Lupinus nanus, L. mutalilis, L. hybridus, L. albus, L. grandiflorus, L. dunetti, Mimosa pudica, and
Acacia lophanta.
Flahault (1878) examined individuals of three hundred and fifty
species of Phanerogams and found that those examined in the genera
Lupinus, Cercis, Gymnocladus, Guilandinia, Acacia, and Mimosa had distinct stelar initials and a common group of initials from which developed cortex, epidermis, and root cap.
Tiegs (1912) x*eported three histogens, plerome, periblem, and
protoderm-columella, in the promeristem of roots of three species of
Leguminosae (Vicia villosa - lateral roots, Pisum sativum, and Trefolium repens). The central cylinder originates from the plerome, the cortex
from the periblem, and the epidermis as well as the root cap from the protoderm-columella initials.
Schliepp (1926), summarizing information on root meristems, 3 distinguished two zones in the root apexs corpus and tunica. The difference between corpus and tunica results from the pattern of division of their cells. By his definition, that part of the root where the number of cell rows increase away from initial zone is called corpus, while the number of cell rows increase towards the initial zone is called tunica. Since Schiiepp’s study dealt only with the pattern of derivation of cells, it is not possible, to correlate it directly with
Hanstain's histogen theory.
‘Neuman (1939) found that in Mimosa and Lupinus the plerome and periblem originate from a central cell. Hie central cylinder is derived from the plerome, the cortex from the periblem. The columella of the root cap originates from columella initials. The peripheral part of the root cap originates by periclinal divisions in the
"dermatogen" according to Neuman.
The only investigation on zonation in the soybean (variety Mammoth
Xellow) root tip which has come to the author's attention is that of
Bell (193U)- Be found that the pattern of development corresponded to that of Janczewski's fourth class of angiosperms, namely, that the stele, cortex, epidermis, and root cap, instead of arising from definite histogens, originate from a common group of meristematic cells. The results of the present study, however, indicate a more complex root apex organization in the soybean. MATERIALS AND METHODS
The primary roots of the Monroe variety of Glycine max ,L. Merrill were used in this study. The plants were cultured in an aerated four-salt solution under controlled illumination and temperature.
Soybean seeds were first soaked for 2k hours in the culture solution. Then* in order to reduce or to eliminate surface contamination* they were treated for five minutes in a dilute disinfectant consisting of one part chlorox (5-25 per cent sodium hypochlorite by weight) to 20 parts water* a treatment which considerably increased the percentage of seeds germinating. The seeds were then placed in ’’germination crocks.u The l|r liter crocks were covered with cotton mosquito netting to support the beans and the culture solution level was maintained slightly above the cotton netting in order to keep the seeds moist.
After 5 days* seedlings, selected on the basis of uniformity of size and length of primary roots, were transferred from the germination crocks to quart mason jars. The jars were painted black on the outside in order to avoid the growth of algae in the culture solution. One seedling was placed in each jar, the root being inserted through a hole in the rubber stopper.
Aerators for both germination crocks and jars were made of £ cm. pieces of porous carbon tubing obtained from National Carbon Co.*
Cleveland, Ohio. One end of the carbon tube was plugged with a rubber stopper* the other end was attached to an 18 cm. length of glass tubing.
These aerators were then connected in parallel to an air line by means of 11T11 tubes. In the germination crocks, the aerator tubes were 5 inserted through the mosquito netting. In the jars, the aerator tubes were inserted through a hole in the rubber stopper used to support the seedlings.
The plants were cultured in a ventilated room under a bank of twelve 96-inch General Electric, T-8, kf?00 white fluorescent tubes.
Eighteen 60-watt incandescent bulbs, nine on each side of the bank of fluorescent tubes, were set up to provide supplementary light in the red end of the spectrum. The bank of lights was adjusted at the top of the plants to give a light intensity of 900-1000 foot candles as measured with Weston Illumination Meter - model 756, and was adjusted occasionally to compensate for lamp ageing. A photoperiod of 15 hours was maintained throughout the experiment. The temperature of the ventilated room was kept at 16-18° C. from 8 P. M. to 5 A. M. (dark hours) and at 22-2h° C. from 5 A. M. to 8 P. M. (light hours).
A culture solution suggested by Meyer (19h5) was prepared from stock solutions of (1) KNO^ (1 Molar), (2) KHgPO^ (l Molar), (3)
Ca(U0^)2 (1 Molar), (U) MgSO^ (1 Molar), (5) a solution containing 5 gm.
FeCl^ and 5 gm. of tartaric acid per liter, and (6) a solution containing 1.5 gm. MnCl2 0.1 gm. ZnClg, 0.05 gm- CuCl2*21^,0, 2.5 gm. H BO , 0.05 gm. Mo0_ per liter. A liter of culture solution 3 3 3 contained 2 cc. each of solutions (l), (2), and (U), 3 cc. of solution
(3), and 1 cc. each of solutions (5) and (6).
The calculated initial osmotic concentration of the culture solution was 0.50 ± 0.03 atmosphere, with an initial pH of U.O ± 0.2.
The culture solution was changed once a week.
The root tips were fixed in a solution of 5 cc. formalin, 5 cc. propionic acid, and 90 cc. of £0 per cent ethyl alcohol, under a reduced pressure of k5>0 mm. of mercury. An ethyl alcohol series was used for dehydrating, and a toluol series for dealcoholation. The root tips were embedded in rubber paraffin and were cut 6 - 7 ^ thick.
The sections were stained with safranin and fast green according to a schedule suggested by Johansen (19lj.O). 7 GENERAL PATTERN OF ZONATION
Applying the terminology of Engard (19UU)j the development of the
primary root of the soybean may be represented schematically as follows:
Fundamental Primary permanent Ilistogens tissues tissues
Stelar initials Procambium----- > Primary xylem, stelar cambium, primary phloem, and pericycle
Promeristem-I Meatic meristem— > Endodermis , cortical. par e nc hyrna , and / hypodermis ^'Common initials Protoderm Epidermis
Columella ---- 1> Root cap
The root promeristem is composed of the following two histogens:
(l) the stelar initials which give rise to the procambium; and (2) the
common initials of the meatic meristem, protoderm, and the columella.
The dividing, enlarging, and elongating cells of the derived tissues
later differentiate into the primary permanent tissues.
1. The Stelar Initials and Their Derivatives
Six to eight cells (as seen in median longitudinal sections)
constituting the stelar initials are situated at the apex of the
central cylinder. They usually appear rectangular, pentagonal, or
hexagonal in both cross and longitudinal median sections. There are
no intercellular spaces in the tissue. The isodiametric cells are
thin-walled, non-vacuolated, and 6 to 12 in diameter.
* The meatic meristem, protoderm, and columella are derived from a
group of initials; this group of initials will be referred to as
c ommon ini tials. The procambium, derived from the stelar initials, is composed of
cells in vertical rows. This tissue consists of densely staining,
slightly longitudinally elongated cells. In cross sections the cells
appear rectangular, pentagonal, or hexagonal. There are no inter
cellular spaces in the tissue.
At a distance of about 200 /x. behind the stelar initials the cells
of the central region of the root enlarge and develop conspicuous
vacuoles. These cells are the forerunners of the central portion of
the primary xylem. At this level, the groups of cells which alternate with the projections of the tetrarch primary xylem differentiate into
primary phloem. Portions of the pericycle adjacent to the primary
xylem ridges are two to three cell layers thick while portions next to
the phloem are one or two cell layers thick. The pericycle is composed
of cells with dense cytoplasm and large nuclei.
The Common Initials and Their Derivatives
In longitudinal section, the common initials of the meatic meristem, protoderm, and columella appear as six to eight tiers of
small non-vacuolated cells which lie between the columella and the
stelar initials. In cross section, the cells appear pentagonal or hexagonal. Forty cells which were measured were 16 to 20 u in diameter and 6 to 10 thick.
The cells of the meatic tissue do not stain as deeply as the procambial cells and are much larger. Small intercellular spaces are characteristic of this tissue. All layers of the meatic tissue are initiated from the peripheral cells of the common initials. ¥/hen this tissue, is initiated, the number of cell layers is 8 to 11 and remains constant until it differentiates into cortical parenchyma. New cells are formed in this tissue by occasional periclinal divisions, but this does not change the initial number of layers. In certain species of plants, Williams (19U7 ) found that the meristematic endodermis acts as a cambium giving rise to all cortical tissue. No such meristematic endodermis is found in the soybean root.
The protoderm, from which the epidermis is derived, is one cell layer wide and is derived from the common initials (Fig. 2). Only anticlinal cell divisions occur in the epidermis, which therefore remains a single layer of cells.
In the root cap, there are two sharply defined regions: the columella and the peripheral part of the root cap. The columella is derived from common initials by transverse cell divisions while the peripheral part of the root cap is derived from the protoderm. The columella consists of regular longitudinal rows of highly vacuolated cells in the central portion of the root cap. In the formation of the peripheral region of the root cap, only the cells of the protoderm divide periclinally. Each row of cells which is derived directly from the protoderm remains a single row of cells (Fig. 2.). This pattern of division is the same as that found in Sinapsis alba (Wagner, 1939).
The peripheral region of the root cap adjacent to the columella is nine to twelve cell layers thick. The number of rows gradually becomes smaller, until, at a distance of 3 Mm- from the root tip, it is one cell layer thick.
2* Comparison of Apices of Primary Roots of Various Ages
The embryo root apex is more massive than the primary root apex of 10
-PC -PD M M /n\ PR CO
Fig. 1. Diagram illustrating the organization and the direction of cell divisions, ci, common initialsj co, columella; mm, meatic meristem; pc, procambium; pd, protoderm; pr, peripheral part of root cap; si, stelar initials. 11
Fig. 2. Longitudinal section of the apex of an embryo root of soybean. 125X. pc, procambium; pd, protoderm. 12
a seedling (Figs. 2, 3 , and b)> though the same general organization is
found in both. The embryo root apex has a diameter of 635yu. (median of
seven roots), while the apex of lb-day-old"* seedlings has a diameter of
357/V. (median of seven roots). The measurements, in both cases, were
taken in a plane through the stelar initials.
Table 1 shows the number and thickness of cell layers in the
various zones of the embryo root and of 3-day, 8-day, and Hi--day
seedling primary roots. The procambium of the embryo root is 28 cells
in diameter (median of 5 roots), as compared to 18 cells (median of 5
roots) in the primary root of lb-day seedlings. The meatic tissue of
the embryo root is thicker than that of the primary root of seedlings,
though the number of cell layers is approximately the same in both
embryo and seedling root. The columella, on the other hand, is shorter
and consists of fewer cell layers in the embryo root apex than in
seedling apices. The length of the columella and thickness of the
peripheral part of the root cap increased gradually with the age of
the root, while the thickness of the meatic tissue and procambium decreased during the time interval covered in these experiments.
This and similar expressions of time will refer to the number of
days following the time when soybean seeds, which had been soaked for
2b hours, were first put into germination crocks. Fig« 3- Longitudinal section of the primary root apex of 3-day soybean plant. lljOX. Fig. L • Longitudinal section of the primary root apex of an
8-day soybean plant. II4OX. Table 1. Diameter of the primary root3 and number and thickness of cell layers of different zones in primary roots of the embryo and seedlings of various ages cultured in aerated solution.
Seedlings linbryo 3-days old 8-days old lh-days old Thickness Thickness Thickness Thickness No. of of cell No. of of cell No. of of cell No. of .of cell cell . layers cell layers cell layers cell layers layers in layers in layers in layers in ^
Procambium'' 2U-30 2li-0-U25 20-28 255-320 18-20 1 7 0 - 2 1 2 18-22 1 2 7 - 1 6 1
Median 28 1*08 26 297 2 0 201* 18 153
Meatic tissue'** 9 - 1 0 212-255 9-10 163-187 8-9 102-127 8-9 85-92
Median 9 2 3 8 9 1 7 0 9 119 9 87
Columella** 13-11* 1 3 6 - 1 7 0 1 5 - 1 6 297-311* 16-19 323-371* 1 7 - 2 2 336-391
Median 13 193 15 297 18 31*0 2 0 371*
Diameter of the primary root* 5 5 2 - 6 8 0 ju. I4.8U—6 1 2 ^ 382-1*67 323-382 p
Median 635 552 1*25 357 M-
* These values represent the range of 7 measurements which were taken in a plane through the stelar initials.
The values in each case represent the range of 5 measurements. Measurements of procambium and meatic tissue were taken at a level of 160 p, behind the stelar initials. Measurements of the columella were taken from the end of the common initials nearest the root tip. 16
DISCUSSION
Eriksson (1878) and Flahault (1878) pointed out that the root apices of some genera of Leguminosae consist of two histogens: the stelar initials and the common initials of cortex, epidermis, and root cap. The same marked zonation was found in the soybean root apex.
The soybean root apex, therefore, does not belong to Janczewski*s fourth angiospermous type as Bell (193^-t) suggested but rather to
Eriksson's fourth type. Eriksson (1878) found two sharply defined regions, the columella and the peripheral region, in the root cap of
Lupinus. In the soybean root cap, the boundary between the columella and the peripheral portion of the root cap are fairly sharp and are obviously of different origin. In Lupinus, Eriksson found that the columella originates from the common initials by transverse cell divisions while the peripheral part originates from the common initials by tangential cell divisions. In the soybean, similar transverse cell divisions were found in the columella while the peripheral portion of the root cap is derived by periclinal divisions of the protoderm.
Brumfield (19U3), by mapping chromosomal rearrangements occurring during mitoses in Viola root cells after treatment with X-rays, was able to show that the abnormalities occurred in cells of segments of the root extending from the center to the periphery. The shape of these segments suggested to Brumfield the possibility that the whole root was derived from three or four "apical cells." The present study has revealed no "apical" or "central" cells in the soybean root such as
Brumfield (±9k3), Neuman (1939), and V. Guttenberg (l9h0, ± 9 h l) have suggested for other Leguminosae. I
Shiiepp (1926) classified the Leguminosae into four categories:
(l) Those in which the corpus forms the central cylinder, while the tunica forms the cortex, epidermis, and root cap. (2) Those in which the corpus forms the central cylinder and inner cortex, while the tunica forms outer cortex, epidermis, and root cap. (3 ) Legumes in which the corpus forms the central cylinder and cortex, while the tunica forms epidermis and root cap. (io.) Legumes in which a common meristematic zone forms the root cap, epidermis, cortex, and central cylinder. Judging by the patterns of cell division within the root meristem, soybean does not fit into any of the categories listed by
Schliepp.
1 18
SUMMARY
1. The primary root apex organization of soybean is not that of
Janczewski’ s fourth angiospermous type, nor does it fit into any of
the categories listed by SchUepp for the Leguminosae. The present
study has revealed no "apical’* or ’’central" cells in the soybean root.
2. The primary root tip of soybean consists of three regions:
the promeristem, the fundamental meristems, and the primary permanent
tissues.
3. The promeristem consists of two histogens: the stelar
initials and the common initials. The procambium is derived from the
stelar initials while the meatic meristem, protoderm, and the columella
of the root cap are derived from a group of common initials.
2*. The root cap consists of two parts: the columella and the
peripheral portion. The columella is directly derived from common
initials while the peripheral part of the root cap is derived from
protoderm.
5. The procambium gives rise to the primary xylem, primary
phloem, cambium, and pericycle. The meatic meristem gives rise to the
endodermis and cortical parenchyma. The epidermis is derived from protoderm.
6. Embryo root apices are more massive than those of seedling primary roots of various ages studied; however, all show the same
general zonation. 19 LITERATURE CITED
Bell, Willis H. Ontogeny of the primary axis of So.ja max. Bot. Gaz.
95: 622-635- 193k-
Brumfield, R. T. Cell lineage studies in root meristems by means of
chromosome rearrangements induced by X-rays. Amer. Jour.
Bot. 30: 101-110. 191+3-
Engard, C. J. Organogenesis in Rubus. Univ. Hawaii, Research
Publication 21. 191+1+ •
Eriksson, J. Ueber das Urmeristem der Dikotylen-Wurzeln. Jb. Wiss.
Bot. 11: 380-1+36. 1878.
Flahault, C. Recherches sur 1'accroissement terminal de la racine
chez les Phanlrogames. Ann. Sci. Wat., Bot. 6: 1-168. 1878.
Guttenberg, H. Van. Der prim&re Bau der Angiospermenwurzel. Linsbauer's
Handbuch der Pflanzenanatomie. Berlin. 191+0.
______- Studien iiber die Entwicklung des Wurzelvegetations-
punktes der Dikotyledonen. Planta 35 s 360-396. 191+7 •
Hanstein, J. V. Die Entwicklung des Keimes der Monokotylen und
Dikotylen. Bot. Abh. Morph, u. Physiol. Bonn. 1: 1-112. 1870. Janczewski, E. De. Recherches sur 1 ’accroissement terminal des racines
dans les Phanerogames. Ann. Sci. Nat., Bot. 20: 162-201.
1871+.
Johansen, D. A. Plant microtechnique. McGraw-Hill Co. Inc., New York.
191+0 .
Meyer, B. S. Effects of deficiencies of certain mineral elements on
the development of Taraxacum kok-sagbyz. Amer. Jour. Bot. 20
32s $23-528. 19U5.
Nttgeli, !. Wachsthumsgeschichte der Laub- und Lebermoose. Zeitschrift
fttr Wissenschaftliche Botanik 2: 138-210. 18U5-
Neumann3 0. \5ber die Bildung der Wurzelhaube bei Juglans, Mimosa,
und Lupinus. Planta 30: 1-20. 1939*
Schuepp, 0. Meristeme. Linsbauer’s Handbuch der PXlanzeanatomie.
Berlin. 1926.
Tiegs, E Beitr&ge zur Kenntnis der Entstehung und des Wachstums der
Wurzelhauben einiger Leguminosen. Jb. Wiss- Bot. 52: 622- 6U6. 1912.
Wagner, Bber die Entwicklungsmechanik der Wurzelhaube und des
Wurzelrippenmeristems. Planta 30s 21-66. 1939. I i
21 j
II. GROWTH AND TISSUE DIFFERENTIATION IN PRIMARY ROOTS
Although there is considerable information available on root structure, there are only a few accounts of tissue differentiation in roots (Esau, ±9h3 j Engard, 19kk» Goodwin and Stepka, 1 Popham,
1914-7Williams, 1914-7J Wetmore, 19h7} and Heimsch, 1951)*
Anatomical studies on the roots of soybean have been few. In their study of the origin of dicotyledonous roots, Van Tieghem and
Douliot (1888) mentioned that the root tip of soybean possesses four vascular strands. The pericycle, according to these authors, is three cell layers thick adjacent to xylem ridges and two cell layers thick adjacent to phloem areas. Secondary roots arise in the pericycle at loci opposite xylem ridges. In a brief investigation of the soybean seedling, Compton (1912) stated that the root contains a tetrarch xylem.
Bell (19314) dealt with ontogenetic changes in the root structure of soybean. He found that the epidermis, endodermis, and pericycle each consists of a single layer of cells. Root hairs arise from epidermis and secondary roots arise from the pericycle. His study gave little attention to the differentiation and maturation of tissues. The present work grew' out of an interest in the time, place of the origin, and direction of tissue differentiation in the primary root of Glycine max
L. Merrill. 22
PROCEDURES
The method of culturing the soybean plants, and the techniques used in the preparation of microscope slides are essentially the same as those described in part I of this dissertation, except for an additional series of non-aerated plants. These plants were cultured in crocks and jars which were without aerators.
Serial cross sections and longitudinal sections of primary roots only were cut 10-13 J\ thick. Free-hand sections of living roots were also studied.
Polychromed methylene blue (Van Fleet, 1950) was used for staining the Casparian strips of the endodermis walls in the free-hand sections.
The reagent was prepared by dissolving 0.25 gm. methylene blue, and
I|. gm. lithium carbonate in 300 cc. of water heated to 90° C. for 10 minutes, after which 5 cc. of acetic acid was added. Phloroglucin and hydrochloric acid were used as a test of lignification. Fresh free-hand sections were placed on a slide in a few drops of a solution of phloroglucin in 50$ alcohol (l;100). After several minutes a drop of 25$ hydrochloric acid was added. A red color indicated the presence of lignin. 23
EXPERIMENTAL RESULTS
1. External Morphology
The root system of the soybean consists of numerous lateral roots
arranged in four longitudinal rows along the primary root. The
lateral roots first appeared at the base of the primary root, i. e.,
just below the hypocotyl. Initiation of lateral root primordia
continued acropetally. No primordia, observable with the naked eye,
ever developed within 3 cm. of the root apex. The roots of soybeans
cultured in aerated solutions differed markedly from those in non-aerated cultures. In the former, the primary roots were much
longer, smaller in diameter, and the secondary roots much more numerous.
The difference in length of primary roots became evident within
four days after soaking the seeds. At the end of li| days the length
of primary roots in aerated solution was 32 cm. (median of 15 roots),
as compared with 9.2 cm. (median of 19 roots) for non-aerated plants.
Over the three-week period of the experiment, the rate of growth of the
primary roots in aerated solutions was slower at first, gradually
increased to a maximum on about the ninth day, after which it
progressively decreased. When the length of the root was plotted
against the age of the seedling a sigmoid curve was obtained. Figure p
illustrates the changes in the total length of the primary roots of
soybeans cultured in aerated solutions, and in non-aerated solutions.
A comparison of the two curves indicates that primary root growth was much retarded when plants were cultured in non-aerated solutions.
Roots of non-aerated plants, though shorter, were greater in 2U
40
z
>- ,Q a: s< 20 c c CL O X H IS uiz
20
AGE OF SEEDLINGS IN DAYS
Fig. 5* Length of primary root of Glycine max seedlings of various ages cultured in aerated and non-aerated solutions. Each dot represents a measurement from 1 plants the encircled dots represent medians. 25 diameter than those of aerated plants. In both aerated and non-aerated seedlings, younger roots were always greater in diameter than older roots. The diameter (l cm. behind the root apex) of primary roots of
5-day seedlings cultured in aerated solution was 0.8 mm., while the corresponding diameter of non-aerated roots was 1.2 mm. In lU-day-old seedlings, root diameters measured 1 cm. behind the apex, were respectively, 0.6 mm. and 0.9 mm. All of these values represent median measurements of 9 roots.
In both aerated and non-aerated seedlings, secondary roots began to appear when plants were 5 days old. The number of secondary roots thereafter increased very rapidly on aerated roots, and only slowly on non-aerated roots (Fig. 6 ).
2_. General Structure of the Primary Root
At 1 cm. behind the root apex, the primary root of Glycine max has a uniseriate epidermis. The cortical parenchyma consists of loosely arranged cells with numerous intercellular spaces, while the stelar tissue is more compact. The cell walls of the endodermis are thickened with Casparian strips. Within the endodermis lies the pericycle, a parenchymatous tissue one to two cell layers thick in the regions adjacent to the primary phloem, and two to three cell layers thick opposite the xylem ridges. The tetrarch primary xylem near the base of a 10-day primary root is well defined with large, thin-walled xylem elements in the center and v/ith smaller, lignified xylem elements in positions nearest the pericycle. Lignification of primary xylem is centripetal (exarch). The primary phloem appears as four strands of cells lying between the primary xylem ridges. median number of roots found on on found of roots number median in aerated and non-aerated solutions. Each point represents the point represents Each solutions. non-aerated and in aerated Rig.
6 NUMBER OF SECONDARY ROOTS Nme fscnayros nsyenpat cultured plants soybean on roots of secondary Number . 5 O G OF AGE 5 9 NON-AERATED aerated seedlings. EDIG I DAYS IN SEEDLINGS 10 15 2 0
26 27
3• Tissue Differentiation
Tissue differentiation in plants may be regarded as the diversified morphological expression of a progression of chemical and physical processes occurring within their cells. A cell or tissue will be considered differentiated when a cell or the cells of the tissue can be distinguished from surrounding cells by differences in chemical properties or physical properties, such as size, shape, inclusions, differences in the cell wall, or number of mitotic figures (popham, 19J47).
A. Primary Phloem
During initial stages of phloem development frequent divisions occurred in incipient phloem regions of the procambium. Phloem strands were recognized by their cell arrangement, cell size, and location.
They were located adjacent to the pericycle, alternating with the xylem ridges and were composed of small cells which were arranged in semi-circular rows. Differentiation of primary phloem areas occurs
203 /if (average of 2p roots) behind the stelar initials in aerated roots and 200 jq (average of 2p roots) behind the stelar initials in non-aerated roots (Table III).
The distance between the stelar initials and the first mature sieve tube element is almost the same for (l) roots which were cultured in aerated solution or in non-aerated solution, and (2) roots of different ages. The level at which the first mature sieve tube element appears in any one of the four phloem areas is U I O ^ (average of 30 roots) behind the stelar initials in aerated roots and i|23 (average of 29 roots) in non-aerated roots. The levels at which a mature sieve tube element first occurs in all four of the phloem areas are I4.67 (average 28 of 29 aerated roots) and U56/K (average of 30 non-aerated roots) behind the stelar initials.(Table III, Fig. 8 ). The level of maturation of three sieve tubes in any one of the four phloem areas is 796
(average of 29 aerated roots) and 698 (average of 30 non-aerated roots) behind the stelar initials. The corresponding levels at -which three mature sieve tubes are seen in each of the four phloem areas are
886 u (average of 27 roots) and 8 13 ^ (average of 31 roots), in aerated and non-aerated roots respectively (Table III, Fig. 9)• This indicated that the aeration did not effect the levels of differentiation and maturation of sieve tube elements.
B. Primary Xylem
The acropetal development of the tetrarch primary xylem involves three fairly distinct processes: l) cell differentiation, evidenced by cell enlargement and elongationj 2 ) cell wall thickening; and 3 )
lignification. Cell enlargement and elongation begins in the center of the xylem and proceeds centrifugally and acropetally. Xylem differentiation occurs at about the same level as primary phloem differentiation (at an average distance of 203 f\ from the stelar initials in 28 aerated plants and 200 in 29 non-aerated plants)
(Fig. 7)- Xylem cell wall thickening and lignification, on the other hand, begins in xylem elements located next to the pericycle and proceeds centripetally and acropetally. The initiation of wall thickening is observable at an average level of 2733 behind the
stelar initials in aerated roots (average of 25 plants) and 27^5 ^ in. non-aerated roots (average of 23 plants) (Table III). The levels at which initial lignification of the xylem cells next to the pericycle Table II. Levels (in cm.) of tissue differentiation and root hair initiation in aerated and non-aerated primary roots of soybean seedlings of different ages.
Age of seedlings in days 5 7 10 lb 21
Aerated root Range'1’ 0.8-1.3 1.5-1.6 1.0 -1 4 0.7-0.75 0.5-0.8 Xylem Median 1.0^ 1,52 1.2 0.72 0.7 lignification Non-aerated Range* 0.8-0.9 0.7-0.8 0.7-0.8 u.65-0.8 0.7-0.82 root . Median 0.86 0.73 0.78 0.7 0.75
Aerated root Range* 2-2.5 1.8-2.2 1.2-1.5 0.8-1.2 0.7-1.0 Casparian Median 2.35 2.0 1 4 0.9 0.8 thickenings Non-aerated Range* 0.8-1.2 0.8-1.2 0.8-1.0 0.5-0.7 0.5-0.7 root Median 1.0 1.0 0.85 0.6 0.6
Aerated root Range* 0.9-1.5 7.2-8.2 18-19.5 21-23 21-23 Root hairs Median 1.2 7.5 19 22 21.5
Non-aerated Range* 04-0.8 04-0.9 0.7-0.9 0.5-1.2 0.9-1.5 root Median 0.6 0.8 0.8 0.9 1.0
Aerated root Range*’ 3.0-3.6 7.5-8.0 17-184 19-22 20-23 Stelar cambium Median 3 4 7.6 17.5 20.5 21.5
Non-aerated Range* no cambium 3.0-3.3 b.2-5.0 ii.6-54 6.0-7.5 root Median no cambium 3.1 R.6 5.0 7.0
* The values represent a range of 5 or more measurements for each age level. All measurements were made from the root tip. Fig. 7* Transverse section of a 7-d.ay aerated root taken 214.0 microns behind the stelar initials. £, pericycle; ph, primary phloem; x* primary xylem. 12 5X. 31
Fig. 8. Transverse section of a 7-day aerated root taken U
Fig. 9- Transverse section of a 7-day aerated root taken 996 microns behind the stelar initia3_s. £, pericycle; s, sieve tube; x, primary :xyleiii. ll£X. Table III. Levels of tissue differentiation (inA\) and lateral root initiation in aerated and non-aerated primary roots of soybean seedlings of different ages.
Aerated roots Won-aerated roots "HO.'"GY" Tissue Range’"’* Median Mean Range** Median Mean no. or plants plants Gap thickness 312-1*20 31*8 359 22 300-14-56 358 356 18
Pericycle differentiation 150-182 160 163 26 150-196 169 168 28
Phloem differentiation 190-221 200 203 28 182-221 198 200 29 CM 0 Xylem differentiation 190-221 200 28 182-221 198 200 29
Saturation (no cytoplasm) of sieve tube elements (a) 360—14-92 422 1*10 30 351-10U I420 1|23 29
(b) Ji90-570 hSS 1*67 29 377-572 hSS 1*56 30
(c) 599-996 ISO 756 29 1*69-936 715 698 30
(d) 636-122U 871 886 27 661-1170 832 813 31
Epidermis differentiation 1170-1323 1261 1255 29 1118-1290 1235 1233 21*
Lateral root initiation 171*0-2028 I8I4.O 1863 26 1760-2093 1859 1871 21
Xylem cell wall thickening 2580-2809 2720 2733 25 2530-286i| 2755 m s 23
* Maturation of sieve tube elements: (a) The level at which the first mature sieve tube elements appears in any one of the four phloem areas. 3k occurs in aerated and in non-aerated roots are shown in Figs. 13 and lij. respectively. It will be noted that in primary roots which grew in non-aerated solution, the level of lignification did not vary too much with the age of the seedling, but remained within a range of 0.7 to
0.86 cm. (each represents a median of 5 roots) from the root apex.
In aerated roots, on the other hand, the level of lignification varied markedly with the age of the seedling. It changed rapidly from a point 0.8 (median of 5 roots) from the root apex in l;-day seedlings to a maximum of 1.7 cm. (median of 5 roots) from the apex in 6-day plants, and then gradually reverted, during ■the next two weeks, to a point about 0.7 cm. (median of 5 roots) from the apex (Figs. 13 and 11;,
Table II). Lignification of the xylem cells in the center of the root was first noted on the 8th day in primary roots cultured in aerated solution, and on the 10th day in those grown in non-aerated solution.
C. Stelar Cambium
Cambium was first noted on the i+th day in primary roots cultured in aerated solution and on the 6th day in those cultured in non-aerated solution. Differentiation of stelar cambium occurs before the primary xylem next to the pericycle has lignified. It first appears in the base of the root in the form of isolated curved sheets of meristem in
Maturation of sieve tube elements (continued): (b) The level at which mature sieve tube elements first occur in each of the phloem areas.
(c) The level of maturation of 3 sieve tube elements in ary one of the four phloem areas.
(d) The level of maturation of 3 sieve tube elements in each of the four phloem areas.
These values represent . . the range of levels of tissue differentiation observed in roots of all plants of all ages studied. 35>
!
c a m
t
Fig. 10. Transverse section of a 6-day aerated root taken 6.3
cm. behind the stelar initials, cam, stelar cambium; _en, endodermis; i p, pericycle; s, sieve tube; x, primary xylem. ll^OX. ! and the levels of stelar cambium initiation. cambium stelar of levels the and Fig. 11. The distance between the basal portion of the roots roots the of portion basal the between distance The 11. Fig.
LEVEL of cambium initiation in cm. 2 O 10 G O SELNS N DAY'S IN SEEDLINGS OF AGE 5 AERATED NON—A E RA T ED 10 15 20 36 Fig. 12. Transverse section of an 8-day non-aerated root taken mm. behind the stelar initials, p, pericycle; s, sieve tube;
secondary root primordium; x, primary xylem. 120X. 38
Fig. 13. Length of primary root (R) and levels of initiation of
stelar cambium (Gam), root hairs (H), Gasparian strips (Cas), and
lignification of xylem cells next to the pericycle (X) in primary
roots of different age seedlings cultured in aerated solutions.
Measurements were taken from the root tip. Each dot represents the median of the initiation levels in $ to 7 plants, except that each dot represents the median length of 15 primary roots (R). LEVEL OF INITIATION IN CM, 40 2
0 G O SELNS N DAYS IN SEEDLINGS OF AGE 10
20 CAS CAM . R 39 I Fig. 1U. Length of primary root (R), and levels of initiation pf stelar cambium (Cam), root hairs (H), Casparian strips (Cas), and lignification of xylem cells next to the pericycle (X) in primary roots of different age seedlings cultured in non-aerated solutions.
Measurements •were taken from the root tip. Each dot represents the median of the initiation levels in 5 to 7 plants, except that each dot represents the median length of 15 primary roots (R). LEVEL of initiation in cm. PO o
cn
O
ro o
t=- H the 'parenchyma on the inner side of each primary phloem strand. From the beginning, it differentiates laterally and acropetally. In aerated roots acropetal differentiation proceeds rapidly, while in non-aerated roots it is much retarded (Table II, Fig. 11). Subsequently pericycle cells opposite the xylem ridges undergo periclinal divisions, forming bridges connecting the sheets of stelar cambium (Fig. 10).
Only one record of the level of stelar cambium formation in the root could be found in the literature, namely, that of Popham (±9k7) in the root of Jatropha cordata. Eiy the end of the third week, the stelar cambium of Jatropha cordata extends to within 1.6 mm. of the root apex. In soybean the level of cambium initiation is 21.5 cm.
(median of 5 aerated roots) and 7 cm. (median of 5 non-aerated roots) behind the root apex (Table III).
D. Pericycle
Pericycle differentiates at a distance of 163 p. (average of 26 aerated roots) and 168 u (average of 28 non-aerated roots) behind the stelar initials (Table III). This tissue forms a cylinder of cells bounded on the outside by the endodermis and on the inside by much smaller cells of the stele. It is two to three cell layers thick adjacent to xylem ridges and one to two cell layers thick adjacent to phloem areas.
At levels of Jj.10 jq (average of 30 aerated plants) and 1*23^
(average of 29 non-aerated plants) behind the stelar initials, the pericycle cells begin to enlarge (Fig. 6). At a distance of 756
(average of 29 aerated plants) and 698 (average of 30 non-aerated plants) behind the stelar initials, pericycle cells opposite the phloem k3
strafids become much larger than those opposite the xylem ridges and
their cytoplasm becomes vacuolated (Fig. 9).
E. Pericycle - Secondary Root Origin
Secondary roots arise in the pericycle at loci directly opposite
the four xylem ridges (Fig. 12). Thus the pattern of the primary
structure of the main root determines the arrangement of the secondary
roots in four longitudinal rows. Radial enlargement of several
pericyclic cells adjacent to a xylem ridge is the first evidence of
secondary root formation. This is followed by tangential and radial
divisions of these pericyclic cells. The cytoplasm of the dividing
secondary root initials is very dense. The primordium continues to
enlarge and elongate, forcing the epidermis and cortical tissues
outward and eventually rupturing them.
F. Endodermis
The endodermis of the soybean root passes through two distinct
developmental stages: the pro-endodermis and the endodermis with
Gasparian strips. These stages can be distinguished from each other
by means of the polychromed methylene blue test. The test shows that
Casparian strips are completely lacking in the pro-endodermis.
Characteristic Casparian strips occur on the thin radial and transverse walls of cells of the endodermis although they never become very thick.
The level at which the Casparian strips appear is higher in
aerated than in non-aerated roots, and in younger than in older roots.
The median level of Casparian strip formation in five 5-day aerated
roots was 2.35 cm. from the tip as compared to a median of 1.0 cm. in
five non-aerated roots of the same age. In lit-day seedlings the corresponding values were 0.9 cm. (median of 5 aerated roots) and 0.6
cm. (median of 5 non-aerated roots) (Table II).
G. Cortical Parenchyma
The cortex is derived from the meatic tissue. Its inner boundary
is distinguishable at the level of endodermis differentiation, and its
outer boundary at the level of epidermis differentiation. Cells of
cortical parenchyma undergo tremendous enlargement and become more or
less circular in cross section. The cortical parenchyma consists of
8-11 layers of large cells with prominent intercellular spaces.
These intercellular spaces are first observable 117 ^ back of the
stelar initials.
H. Epidermis and Root Hairs
The epidermis consists of a single layer of cells derived from
the protoderm. All cell divisions in the epidermis are anticlinal.
The epidermis differentiates at an average distance of 122$ behind
the stelar initials in aerated roots (average of 29 plants) and 123 3
in non-aerated roots (average of 21). plants). Bell (193U) stated that the epidermis of soybean develops root hairs early in ontogeny. The present study confirmed this observation. During the first four days
of culturing, the epidermal cells of the soybean root became vacuolated,
elongated, and enlarged. Root hairs differentiated on the fourth day.
No data have been found in the literature on the level of
initiation of root hairs forming on roots cultured under different
environmental conditions. Root hairs of primaiy roots of soybean cultured in aerated solutions were sparsely developed except for
certain regions at the base of the root. . Roots cultured in non-aerated h5 solution produced an abundance of root hairs at levels approximately
0.6 cm. (median of five 5-day roots) and 1 cm. (median of five 21-day roots) behind the root tip (Figs. 13 and lij., Table II). The more luxuriant growth of root hairs in the non-aerated solution corraborates a similar observation made by Snow (1
I. Secondary Tissues
Proceeding from the region of cambium differentiation toward the root base, one finds a progressive increase in the number of secondary xylem elements. Extensive areas of secondary xylem are formed between the primary xylem ridges. The primary phloem tissues and the cells of the pericycle are pushed outward against the surrounding cortical tissue, resulting in the crushing of the primary phloem. At the base of the root, most of the phloem is secondary. The secondary phloem consists mainly of sieve tubes, companion cells, a few parenchyma cells, and phloem fibers.
Phellogen was not found in the soybean root.
J. Root Gap
The average root cap thickness of 22 aerated roots is 359 / \ and the average root cap thickness of 18 non-aerated roots is 356 j\. Those cells in the central portion of the root cap are derived from the common initials directly, while those in the peripheral portion are derived from the protoderm. The thin-walled, vacuolated cells of the central part of the root cap are hexagonal in cross section and pentagonal or rectangular in the peripheral region. There are no intercellular spaces in the root cap. h6
K. Comparison of Growth Region and Tissue Differentiation
in Aerated and Non-aerated Roots
The levels of differentiation of pericycle, primary xylem, primaiy phloem, and epidermis, as well as maturation of sieve tube elements and xylem cell wall thickening, were similar (l) for both aerated and non-aerated roots and (2) for roots of different age seedlings (Table III). This similarity also exists for the level of initiation of secondary roots (Table III). The levels of initiation of xylem lignification, Casparian thickenings, root hairs, and stelar cambium differ not only (l) from aerated to non-aerated roots, but also (2) from young to old seedling roots as shown in Table II.
Figures 13 and llj. represent the levels of differentiation of the various tissues in aerated and in non-aerated soybean roots. k7
DISCUSSION
Soybean roots cultured in aerated solution have a strikingly different growth habit than those cultured in the same but non-aerated medium. In this way they resemble the roots of barley (Bryant, 193U)*
The pattern of differentiation and the direction of development of tissues in the primary roots of soybeans cultured in aerated solution are similar to those of roots cultured in non-aerated solution
Bryant (I93i|) found a considerable difference between the cortical parenchyma of aerated and that of non-aerated barley roots. ' The cortical parenchyma of the former consists of uniformly compact cells with no conspicuous intercellular spaces, while that of the latter is composed of large air passages separated by narrow strands of cells.
In the present work, no difference in the size of intercellular spaces in cortical parenchyma was noted.
Current botanical texts misuse terminology when they divide the root into four regions: root capj zone of cell division^ zone of cell elongationj zone of differentiation and maturation. Differentiation and maturation of various types of cells and tissues occur simultaneously at different levels within the root. Cell enlargement and elongation start at a level very close to the root apex where cell division is still taking place. Goodwin and Stepka (19h5) have called attention to these facts in their study of Phleum pratense. The present study suggests that the old system of zonation of root tissues, based on static concepts, should be replaced by one giving due consideration to the dynamic aspects of differentiation and development
When primary roots grow in non-aerated solutions, not only growth U8 in length but also cambium differentiation is retarded. In both aerated and non-aerated roots, curves for the level of lignification of xylem elements and appearance of Gasparian thickening closely approximate each other. This might lead one to suppose that the processes involved in lignification are correlated with those responsible for Gasparian thickenings, but since our knowledge of the biochemistry of these processes is incomplete, there is little reason at present for concluding that the lignification of xylem and the level of appearance of Casparian thickenings are causally related.
Bell (193-U) stated: "The primary xylem is exarch in its development, the first cells of the proto>ylem differentiating from the procambium at four points adjacent to the pericycle." From his drawings, however, it would appear that his statement refers not to the differentiation of the primary xylem nearest the pericycle, but rather to lignification. Many workers have confused the terms
"differentiation" and "lignification." In soybean, xylem differentia tion begins in the elements located nearest the center of the root and proceeds centrifugally, whereas lignification begins in the xylem elements located next to the pericycle and proceeds centripetally.
Esau (1936), in her study of vessel development in celery, defines a mature vessel as one in which the protoplast and the end wall have disappeared. In the soybean root, the end walls of all primary xylem elements appear to be present when the roots were lb days old. U9 SUMMARY
1* The root system of Glycine max consists of a main tap root with numerous lateral roots, which are arranged in four longitudinal rows.
2. Primary roots of soybean which are cultured in an aerated solution are several times as long as those cultured in a non-aerated solution, and the secondary roots are more numerous.
3- Root hairs of aerated primary roots are sparsely developed except along the basal portion of the root. Roots cultured in non—aerated solution produced an abundance of root hairs at a level of 0.8 cm. behind the root cap.
The levels of differentiation of pericycle, primary xylem, primary phloem, and epidermis, as well as xylem cell wall thickening and maturation of sieve tube elements, were similar and always below the level of Casparian thickenings (1) for both aerated and non-aerated roots and (2) for roots of different age seedlings. This similarity also exists for the level of initiation of secondary roots.
5. In aerated roots, the levels of cambium and root hair initiation were above the level of initiation of Gasparian thickenings.
In non-aerated roots, only cambium initiation was always above the level of Gasparian thickenings. In seedling roots less than 12 days old, the levels of root hair initiation and xylem lignification were below the level of Gasparian thickenings and in the seedling roots more than 12 days old, they were above that level.
6. Cell enlargement and elongation begins in the center of the xylem and proceeds centrifugally. Cell wall thickening and lignifiea- 50 tion, on the other hand, begins in xylem elements located next to the pericycle and proceeds centripetally.
7- Cambium appears first at the innermost edge of the primary phloem strands and develops laterally and acropetally. Cambium was first observed during the fourth day in primary roots cultured in aerated solution and on the sixth day in those cultured in non-aerated solution.
8. Secondary roots arise in the pericycle at loci directly outside the ridges of the tetrarch xylem. 51 LITERATURE CITED
Bell, Willis H. Ontogeny of the primary axis of So.ja max. Bot. Gaz.
95: 622-635- 193 A-
Bryant, A. E. Comparison of anatomical and histological differences
between roots of barley grown in aerated and non-aerated
culture solutions. Plant Physiol. 9: 389-391. 193A-
Compton, R. H. An investigation of the seedling structure in the
Leguminosae. Jour. Linn. Soc. Bot. Al: 1-119- 1912.
Engard, C. J. Organogenesis in Rubus. University of Hawaii Research
Publication No. 21 . 19 AA-
Esau, K. Vessel development in celery. Hilgardia 1 0 : A77—A8 8 . 1936.
_ . Vascular differentiation in the pear root. Hilgardia 15:
299-311. 19 A3-
Goodwin, R. H., and W. Stepka. Growth and differentiation in the root
tip of Phleum pratense. Amer. Jour. Bot. 32: 36-I4.6 . 19A5-
Heimsch, Charles. Development of vascular tissues in barley roots.
Amer. Jour. Bot. 38: 523-537. 1951.
Popham, R. A. Developmental anatomy of seedling of Jatropha cordata.
Ohio Jour. Sci. 5l: 1-20. 19A7.
Snow, L. The development of root hairs. Bot. Gaz. AO: 12-A8. 1905.
Van Fleet, D. S. A comparison of histochemical and anatomical
characteristics of the hypodermis with the endodermis in
vascular plants. Amer. Jour. Bot. 37: 721-725- 1950.
Van Tieghem, Ph. et H. Douliot. Recherches comparatives sur l'origine
des membres Endog^nes dans les plants vasculaires. Ann. Sci.
Nat. Bot., VIIe ser., 8 : 1-660. 1888. 52
Wetmore, R. H. The diff erentiati on of primary vascular tissues in
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AUTOBIOGRAPHY
I, Chao Nien Sun, was born at Tinghsein, Hopeh, China on Nov. 1+,
19H+. I received my high school diploma from the Eighth Normal School
of Hopeh, Chengting, Hopeh, China. My undergraduate work was done at
The National University of Peking, Peking, China, with the degree of
Bachelor of Science in Biology granted in 191+0. I entered the
University of Oklahoma., Norman, Oklahoma, in 1948, from which I received the degree of Master of Science in 1950. From the autumn quarter of 1950 to the spring quarter of 1953, I was enrolled at The
Ohio State University as a candidate for a Ph. D. degree in Botany.
From 191+0 to 191+5, 1 was an assistant and research assistant, and from 191+5 to 191+8, I was a lecturer in the Botany Department, The
National University of Peking. I was a graduate assistant in the
Department of Plant Sciences of the University of Oklahoma during the years 191+8 to 1950. I held the same position in the Department of
Botany and Plant Pathology of The Ohio State University during the year 1950 to 1951* In the autumn of 1952 I accepted a research fellowship in the Institute of Biophysics, St. Louis University, St.
Louis, Missouri, which I held to March, 1953*